University of Virginia Patent Foundation

Weihan Wang

Donald F Hunt

Lichao Zhang

The application discloses compositions, methods, systems, and apparatuses for rapid sequence analysis of proteins, including location of post-translational modifications and disulfide bonds. Limited digestion of fully denatured antibody occurs in seconds by flowing sample in 8M urea at constant pressure through a micro column reactor containing immobilized aspergillopepsin I, resulting in a product mixture containing 3-10 kDa peptides, which is then fractionated by capillary column chromatography and analyzed by both electron transfer dissociation (ETD) and collision activated dissociation mass spectrometry. This method provides 95% sequence coverage of a mAb and detects numerous post-translational modifications. For disulfide bond location, native mAb is subjected to longer digestion times. Release of disulfide containing peptides from accessible regions of the folded protein occurs with short digestion times. The identity of peptides connected by a disulfide bond is determined using ETD and ion-ion proton transfer chemistry.

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Charlottesville, VA, United States of America

Weihan Wang